The long term objective of this work is to reduce air pollution and acid precipitation resulting from combustion of sulfur contaminants in fuels, especially pyrite, the major inorganic sulfur compound in coal. Air pollution is a factor in respiratory disease, and contributes to stress and deterioration of health in general. Acid rain and runoff not only damages the environment, both natural and manmade, but can adversely affect domestic water supplies by increasing solubilization of metals. Pilot studies have shown that microbial desulfurization of coal is feasible and efficient. Genetic manipulation of the microbes involved can increase the process rate enough to render it economical and hence adoptable by industry. The specific aims of this proposal are to establish a system of genetic recombination in thiobacilli, the largest group of sulfur-oxidizing bacteria, in order to identify and manipulate genes controlling sulfur metabolism. In particular, a Thiobacillus plasmid marked with an antibiotic resistance gene will be derived from a large indigenous Thiobacillus plasmid in T. versutus by molecular genetic analysis of Thiobacillus exconjugants having received a transposon carrying such a marker in conjugation with an Escherichia coli donor strain. T. versutus was chosen for its single large plasmid and ability to grow well in conditions compatible with vigorous growth and conjugation by E. coli. The marked plasmid will be identified by standard molecular techniques of gel electrophoresis, Southern blotting, and hybridization. The marked plasmid will then be used in conjugation and transformation experiments with both thiobacilli and E. coli to measure the feasibility and parameters of these genetic processes. As a usable system of genetic transfer is developed, investigation of the genetic basis of sulfur metabolism in T. versutus will be initiated.